PROJECT SUMMARY Alcohol use disorder (AUD) is a prevalent, chronically relapsing disorder in which stress plays a major role. Understanding the neurobiological underpinnings of interactions between stress and alcohol drinking is vital to combating the development and maintenance of AUD. The central amygdala (CeA) plays an important role in regulating alcohol-related behaviors, especially in the context of alcohol dependence, and is responsive to stress. Similarly, the insular cortex (IC) has been shown to regulate craving and seeking behaviors with drugs of abuse, including alcohol, and is also implicated in emotional processing. Dynorphin/kappa opioid receptor (Dyn/KOR) signaling has been shown to encode negative affect, like that which is caused during stress and alcohol withdrawal, and Dyn/KOR are robustly expressed in both the IC and CeA. Furthermore, disruption of Dyn/KOR signaling in the CeA has been shown to block stress-enhanced alcohol drinking in alcohol dependent mice. We have preliminary evidence that the IC sends a large Dyn-expressing (Dyn+) projection to the CeA that may mediate this effect. To our knowledge, no studies to date have examined this ICDyn+?CeA circuit and how it regulates excessive alcohol drinking. Thus, the proposed studies will characterize this circuit, examine how chronic exposure to stress and alcohol affects the circuit's activity, and how manipulation of this circuit affects stress-enhanced alcohol drinking in alcohol dependent mice. Our overarching hypothesis is that the ICDyn+?CeA circuit is a critical mediator of stress-enhanced alcohol drinking in the context of dependence. To test this hypothesis, we will use the Stress-CIE Drinking model, a mouse model that combines chronic intermittent ethanol (CIE) exposure with chronic forced swim stress (FSS). This model produces alcohol dependence and enhanced alcohol intake with stress. For Aim 1, we will characterize the activity of the ICDyn+?CeA circuit in the Stress-CIE Drinking model using viral tracing and immunofluorescence techniques. For Aim 2, fiber photometry will be used to measure ICDyn+?CeA circuit activity during home cage alcohol drinking in the Stress-CIE Drinking model. For Aim 3, will use the Stress-CIE Drinking model combined with designer receptors exclusively activated by designer drugs (DREADDs) to manipulate the ICDyn+?CeA circuit during home cage alcohol drinking. Collectively, these studies will shed light on the role of the understudied ICDyn+?CeA circuit and how it regulates alcohol drinking in a model of alcohol dependence and alcohol-stress interactions.